Calmodulin Binding Domains in Critical Risk Proteins Involved in Neurodegeneration
Round 1
Reviewer 1 Report
This work aims to gain additional insight into the Calmodulin (CaM) Hypothesis in major neurodegenerative diseases, which the author has proposed in previous publications as an extension of calcium dysregulation (Calcium Hypothesis) in Alzheimer’s disease (AD). To this end, the author analyzes the presence of canonical and IQ and IQ-like motifs that mediate calcium-independent CaM binding by visual scanning of protein sequences in several risk factors (proteins) of Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTD), Huntington’s disease (HD), Parkinson’s disease (PD) and Dementia with Lewy Bodies (LBD). The author is aware of the limitations of this methodological approach and states in the Results section that the proposed CaM-binding sequences and motifs in these proteins require experimental validation. Although this work aims to open new perspectives for the search for CaM-dependent mechanisms of calcium dysregulation in major neurodegenerative diseases, in its present form this manuscript looks somewhat speculative. Several points should be addressed in this manuscript before I can recommend it for publication, see below.
1. In the Abstract and in the Introduction sections it should be stated that this article search for canonical and IQ or IQ-like domains that bind to apo-CaM in selected risk factors (proteins). In the Introduction it should be briefly commented the expected molar fraction of apo-CaM in neurons and its relevance for neuronal functions.
2. Taking into account point 1 above, it is recommended to explicitly indicate in the Figure 1 which of the experimentally validated CaMBPs have been shown to bind to apo-CaM, to CaM saturated by Ca2+ or to both conformations of CaM. Also, to make the central hypothesis of this article less speculative the reported dissociation constants of their complexes should be included, to allow for a critical evaluation of the probability of its occurrence in vivo. Finally, since there are many CaMBPs competing for the binding of CaM in neurons, a comment is needed on the putative correlation between known dissociation constants and the type and number of canonical and/or IQ or IQ-like domains found in this work.
Minor point:
Please, normalize the use of the abbreviation of Dementia with Lewy Bodies (LBD) throughout the text. For example, DBL is used instead of LBD on pages 4 (line 104) and 7 (line 223).
Author Response
REVIEWER 1
Note: changes to the manuscript are indicated as blue text.
Reviewer 1.
- In the Abstract and in the Introduction sections it should be stated that this article search for canonical and IQ or IQ-like domains that bind to apo-CaM in selected risk factors (proteins).
Author Response. The term “calmodulin binding domains” is an unbiased term that covers both “canonical and IQ or IQ-like domains” as well as calcium-dependent CaMBPs, so we have not made the suggested change. What’s more IQ motifs were a minor unexpected result and putting a reference to them in the Abstract or Introduction would be misleading. However, since a few IQ/IQ-like motifs were discovered, IQ/IQ-like motifs and apo-CaM were covered in the Discussion.
Reviewer 1. In the Introduction it should be briefly commented the expected molar fraction of apo-CaM in neurons and its relevance for neuronal functions.
Author Response. Again, the primary focus and results is on calcium-dependent CaMBPs with only a few proteins of close to 2 dozen proteins analyzed showing an IQ or IQ-like motif. So, making apo-CaM a central theme would be misleading to the reader.
Reviewer 1. Taking into account point 1 above, it is recommended to explicitly indicate in the Figure 1 which of the experimentally validated CaMBPs have been shown to bind to apo-CaM, to CaM saturated by Ca2+ or to both conformations of CaM. Also, to make the central hypothesis of this article less speculative the reported dissociation constants of their complexes should be included, to allow for a critical evaluation of the probability of its occurrence in vivo. Finally, since there are many CaMBPs competing for the binding of CaM in neurons, a comment is needed on the putative correlation between known dissociation constants and the type and number of canonical and/or IQ or IQ-like domains found in this work.
Author Response. While the reviewer poses some interesting questions and offers some unexplored ideas, these are not in keeping with the focus of the manuscript. This is a brief report with a focus: CaMBPs and potential CaMBPs that are linked to neurodegeneration. The results support the idea that calmodulin and its binding proteins lie at the heart of multiple neurodegenerative diseases and should be the focus of future research. What’s more, the requested data does not exist in the current literature. Until the specific CaMBPs and CaM/apo-CaM are quantified and localized in normal and diseased brains then the issue of protein interactions and dissociation constants cannot legitimately be introduced or discussed. It is much too early for that conversation.
Reviewer 1. Please, normalize the use of the abbreviation of Dementia with Lewy Bodies (LBD) throughout the text. For example, DBL is used instead of LBD on pages 4 (line 104) and 7 (line 223).
Author Response. Four corrections have been made and LBD is now used consistently.
Reviewer 2 Report
The manuscript titled "Calmodulin binding domains in critical risk proteins involved in neurodegeneration" by O’Day is a review on the relationship between calmodulin binding proteins and neurodegenerative disorder.
In my opinion the author gives a good light of the relationship between CaM and critical protein involved in the neurodegeneration.
Author Response
REVIEWER 2
Note: changes to the manuscript are indicated as blue text.
Reviewer 2.
The manuscript titled "Calmodulin binding domains in critical risk proteins involved in neurodegeneration" by O’Day is a review on the relationship between calmodulin binding proteins and neurodegenerative disorder.
In my opinion the author gives a good light of the relationship between CaM and critical protein involved in the neurodegeneration.
Author Response: Clearly this reviewer was satisfied with the manuscript as it was submitted.
Reviewer 3 Report
CIMB-2024427-PEER-REVIEW-V1
The manuscript by Danton H. O'Day, titled "Calmodulin binding domains in critical risk proteins involved in neurodegeneration" analyzes the role of calmodulin in neurodegeneration looking for the presence of calmodulin binding domains in key proteins involved in neurodegenerative disease development. Here, biomarker proteins associated with other neurodegenerative diseases are scanned for the presence of calmodulin binding domains and binding motifs. In most of the analyzed proteins, several CaMBDs-binding domains have been identified, showing how the interaction with calmodulin could be possible for several key proteins in the development of these diseases, confirming calmodulin's role in neurodegeneration.
Overall, the manuscript presents some interesting and novelty points, but some issues should be revised and reconsidered, before the publication of the manuscript in Current Issues in Molecular Biology.
Major points:
In the introduction it is not clear which proteins will be analyzed and which not and it is not clear the reason why some proteins that have been already experimentally validated are here analyzed for the CaMBDs. Please, clarify.
In the results section 2.1 the author declares that LRRK2 has a CaMBD at position 776-795 with 4 binding motifs, one should be 1-14 and not 1-4. Then, the author should specify the sequence of the identified binding domain when referring to CaMBD of PARK7.
In section 2.2 the author affirms that the CaMBD of TMEM175 is present in the cytoplasmatic region but it is not clear how this information has been retrieved. Furthermore, the calcium-independent IQ motif 204LQLAQRPVSLLASP217 is not present in Table 1.
In section 2.3 it is mentioned that SQSTM1 has no presumptive CaMBDs, but in Table 2 there is its IQ-Like Motif. The author should explain it. Furthermore, the FUS CaMBD is written as 301VADYFKQIGIIKTNKKTGQ319 but in Table 2 is 301-309. Please, also for TBK1 and C9orf72 report their CaMBDs sequences.
In section 2.4 TGM1 CaMBD (607RRTVKLHLYLSVTFYTGVS625) showed 2 motifs, the second one is 1-5-10 and not 15-10. Please, modify it.
In the discussion, the author report that: "In addition, three other risk or biomarker proteins (PARK7, LRRK2, PINK1) possess potential binding domains. PARK7 has two binding domains each with multiple binding motifs while LRRK2 has a single identified CaMBD with multiple binding motifs. PINK1 appears to possess a non-canonical CaMBD". However, referring at Table 1, it seems that PARK7 has a non-canonical CaMBD, while PINK1 has two binding domains. The author should clarify this aspect.
Also, "While the Calmodulin Target Database failed to reveal a CaMBD in this region a visual analysis of this basic amino acid rich region (118KGFGGAMTDAAALNILALSP127) revealed 4 binding motifs (Table 1). This suggests GBA may have two independent CaMBDs". However, in Table 1 there is no sequence between 118 and 127 for any protein; please, clarify the positions. Afterward, it seems that CHCHD10 has at least one calcium-dependent CaMBD ("TDP-43, FUS, TBK1, 247 CHCHD10, and C9orf72 all were found to contain at least one calcium-dependent CaMBD with one or more binding motifs") but in section 2.3 is reported that CHCHD10 and SQSTM1 have no presumptive CaMBDs. The author should clarify this aspect. Finally, at the end of page 8 is reported that "TGM3 also has a single CaMBD (493LAVGKEVNLVLLLKNLSRDT512) but with 5 binding motifs" but this information is already mentioned exactly with the same words in this same section (line 259). Please, modify.
Minor points:
At the end of the introduction, after "Along with FUS and TDP-43, SOD1 contributes to inclusions in ALS neurons", add references.
In the discussion after "With a diverse symptomology and occurring in less than 5% of dementia cases, FTD can be inherited but more often spontaneous forms of the disease occur" add references.
Author Response
REVIEWER 3
Note: changes to the manuscript are indicated as blue text.
Reviewer 3. In the introduction it is not clear which proteins will be analyzed and which not and it is not clear the reason why some proteins that have been already experimentally validated are here analyzed for the CaMBDs. Please, clarify.
Author Response: The final paragraph of the Introduction was completely revised to make this point clear.
Original Para:
“To gain additional insight into the role of calmodulin in neurodegeneration, a number of recently identified risk factors were analyzed for their potential ability to bind to CaM. Five proteins have been cited as risk factors for LBD, some of which are also linked to more than one neurodegenerative disease: BIN1 (also AD), TMEM175 (Transmembrane protein 175; also PD), SNCA (also PD), APOE (also AD) and GBA (glucocerebrosidase, a lysosomal enzyme; also PD) [20]. Parkin, PINK1, LRRK2 and PARK7 have been categorized as risk and/or marker proteins for PD [21,22]. Marker and risk genes linked to FTD which encode proteins that accumulate in the frontal and temporal lobes, include tau, progranulin (GRN), FUS and TPD-43.Risk genes that overlap between FTD and ALS have also been revealed including TDP-43 (TARDBP), SQSTM1, VCP, FUS, TBK1, CHCHD10, and C9orf72 [23,24]. Along with FUS and TDP-43, SOD1 contributes to inclusions in ALS neurons.”
Revised Para:
“To gain additional insight into the role of calmodulin in neurodegeneration, a number of recently identified risk factors were analyzed for their potential ability to bind to CaM. Two proteins cited as risk factors for LBD, were evaluated: TMEM175 (Transmembrane protein 175; also PD), and GBA (glucocerebrosidase, a lysosomal enzyme; also PD) [20]. SNCA, Parkin, PINK1, LRRK2 and PARK7 that have been categorized as risk and/or marker proteins for PD were analyzed for their potential CaM-binding ability [21,22]. FUS and TPD-43 (TARDBP), marker and risk proteins linked to FTD, that accumulate in the frontal and temporal lobes, were evaluated as well as risk proteins that overlap between FTD and ALS including SQSTM1, VCP, TBK1, CHCHD10, and C9orf72 and progranulin (GRN) [23,24]. Along with FUS and TDP-43, SOD1 which contributes to inclusions in ALS neurons was also evaluated [23]. The risk proteins for HD that were analyzed were huntingtin and TGMs 1-3.”
Reviewer 3. In the results section 2.1 the author declares that LRRK2 has a CaMBD at position 776-795 with 4 binding motifs, one should be 1-14 and not 1-4. Then, the author should specify the sequence of the identified binding domain when referring to CaMBD of PARK7.
Author Response: 1-4 was corrected to 1-14; the following CaMBD of PARK7was inserted on line 97 (93KEQENRKGLIAAICAGPT110) to correct the omission.
Reviewer 3. In section 2.2 the author affirms that the CaMBD of TMEM175 is present in the cytoplasmatic region but it is not clear how this information has been retrieved. Furthermore, the calcium-independent IQ motif 204LQLAQRPVSLLASP217 is not present in Table 1.
Author Response: The cytoplasmic localization is explained by the addition of “that Uniprot reveals” to the sentence. The IQ motif 204LQLAQRPVSLLASP217 has been inserted into the revised Table
Reviewer 3. In section 2.3 it is mentioned that SQSTM1 has no presumptive CaMBDs, but in Table 2 there is its IQ-Like Motif. The author should explain it. Furthermore, the FUS CaMBD is written as 301VADYFKQIGIIKTNKKTGQ319 but in Table 2 is 301-309. Please, also for TBK1 and C9orf72 report their CaMBDs sequences.
Author Response: The IQ motif issue has been corrected by changing the sentence l. 120-122, “Two proteins (SQSTM1, sequestosome-1; CHCHD10, coiled-coil-helix-coiled-coil-helix domain-containing protein 10) have no presumptive CaMBDs.” to “While SQSTM1 (sequestosome-1) and CHCHD10 (coiled-coil-helix-coiled-coil-helix domain-containing protein 10) have no presumptive calcium-dependent CaMBDs, SQSTM1 possesses an IQ motif.” Table 2 has been corrected (309 changed to 319). The sequences for TBK1 and C90rf72 have been inserted in the text.
Reviewer 3. In section 2.4 TGM1 CaMBD (607RRTVKLHLYLSVTFYTGVS625) showed 2 motifs, the second one is 1-5-10 and not 15-10. Please, modify it.
Author Response: correction done
Reviewer 3. In the discussion, the author report that: "In addition, three other risk or biomarker proteins (PARK7, LRRK2, PINK1) possess potential binding domains. PARK7 has two binding domains each with multiple binding motifs while LRRK2 has a single identified CaMBD with multiple binding motifs. PINK1 appears to possess a non-canonical CaMBD". However, referring at Table 1, it seems that PARK7 has a non-canonical CaMBD, while PINK1 has two binding domains. The author should clarify this aspect.
Author Response: The sentence has been rewritten to reflect the data, “In addition, three other risk or biomarker proteins (PARK7, LRRK2, PINK1) possess potential binding domains. PINK1 has two binding domains each with multiple binding motifs while LRRK2 has a single identified CaMBD with multiple binding motifs. PARK7 appears to possess a non-canonical CaMBD.”
Reviewer 3. Also, "While the Calmodulin Target Database failed to reveal a CaMBD in this region a visual analysis of this basic amino acid rich region (118KGFGGAMTDAAALNILALSP127) revealed 4 binding motifs (Table 1). This suggests GBA may have two independent CaMBDs". However, in Table 1 there is no sequence between 118 and 127 for any protein; please, clarify the positions.
Author Response:
The information was poorly expressed, the sentence refers to a visual analysis of the region that did not involve using the Calmodulin Target Database. The original sentence “While the Calmodulin Target Database failed to reveal a CaMBD in this region a visual analysis of this basic amino acid rich region (118KGFGGAMTDAAALNILALSP127) revealed 4 binding motifs (Table 1). This suggests GBA may have two independent CaMBDs.” was corrected to (l, 236-240) “The Calmodulin Target Database failed to reveal a CaMBD in this region, but a visual scanning of this basic amino acid rich region revealed a potential CaMBD (118KGFGGAMTDAAALNILALSP127) with 4 binding motifs. This suggests GBA may have three CaMBDs of which only two were detected by database analysis.”.
Reviewer 3. Afterward, it seems that CHCHD10 has at least one calcium-dependent CaMBD ("TDP-43, FUS, TBK1, 247 CHCHD10, and C9orf72 all were found to contain at least one calcium-dependent CaMBD with one or more binding motifs") but in section 2.3 is reported that CHCHD10 and SQSTM1 have no presumptive CaMBDs. The author should clarify this aspect. Finally, at the end of page 8 is reported that "TGM3 also has a single CaMBD (493LAVGKEVNLVLLLKNLSRDT512) but with 5 binding motifs" but this information is already mentioned exactly with the same words in this same section (line 259). Please, modify.
Author Response: The reviewer is correct, the reference to CHCHD10 has been removed from the sentence. The duplicated sentence at line 260 was removed.
Reviewer 3. At the end of the introduction, after "Along with FUS and TDP-43, SOD1 contributes to inclusions in ALS neurons", add references.
Author Response: As indicated above a reference [23] has been added at the end of the sentence.
Reviewer 3. In the discussion after "With a diverse symptomology and occurring in less than 5% of dementia cases, FTD can be inherited but more often spontaneous forms of the disease occur" add references.
Author Response: this has been done
Author Final Comment re: Reviewer 3. This reviewer has helped improve the manuscript significantly. The careful reading and specific recommendations facilitated making the revisions and made the author aware of issues to avoid in the future. Thank you.
Round 2
Reviewer 3 Report
The authors have improved the manuscript following the suggestions. Thus, the manuscript is now suitable for publication.
